plasma derived apolipoprotein e  (Athens Research)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on apolipoproteins was assayed using NHS and C1q-depleted human serum (ΔC1q). (A-D) C4b-, C3b-, C1q-, and C9 deposition from NHS, respectively using classical complement deposition conditions (DGVB++ buffer), on coated proteins h.a. IgG (2.5 μg/mL in A, B, D; 1 μg/mL in C), ApoE (10 μg/mL), and negative control A1AT (10 μg/mL). (E) C4b deposition from 2% NHS, 2% ΔC1q, and 2% C1q-reconstituted serum (ΔC1q + C1q, 3.2 μg/ml, based on 160 μg/ml in 100% serum), on coated proteins h.a. IgG (1.0 μg/mL), ApoE (10 μg/mL), and A1AT (10 μg/mL). (F) C1q and C1 binding to immobilized ApoE (10 μg/mL), h.a. IgG (0.5 μg/mL), and A1AT (10 μg/mL), detected using specific antibodies. (G) Fluorescently labeled ApoE (AF488) binding to immobilized C1q (10 μg/mL), and A1AT (10 μg/mL). (H) Fluorescently labeled ApoE (AF488) binding to full C1q protein, and isolated head-, and stalk domains of C1q (all 10 μg/mL). Mean ± SD (A-D, F, G) or mean + SD (E and H) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Dunnett’s multiple-comparisons test (A-D, F), two-way ANOVA with Tukey’s multiple-comparisons test (E), two-way ANOVA with Sidak’s multiple-comparisons test (G), and one-way ANOVA with Dunnett’s multiple-comparisons test (H) were applied to determine statistically significant differences compared to control (A1AT), and per coated protein to compare C4b deposition from NHS, C1q-deficient serum, and C1q-replenished serum (E). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NHS, normal human serum; h.a. IgG, heat-aggregated IgG; ApoE, recombinant human Apolipoprotein E (ε3 isoform from Novoprotein, AF488-labeled protein in G, 2 μg/mL in H); ApoB, Apolipoprotein B; ApoD, apolipoprotein D; A1AT, alpha-1-antitrypsin; RFU, relative fluorescence units.

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Negative Control, Binding Assay, Labeling, Isolation, Control, Recombinant, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on ApoE was assayed using NHS or Factor B (FB)-depleted serum (ΔFB). (A) C3b deposition using alternative complement deposition conditions (Mg2+-EGTA buffer). (B) C3b deposition from 3% sera: NHS, ΔFB, and FB-reconstituted serum (ΔFB + 6 μg/mL FB, based on 200 μg/mL in 100% serum). Mean ± SD (A) or mean + SD (B) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s (A) or Dunnett’s (B) multiple-comparisons test were applied to determine statistically significant differences. **p<0.01, ****p<0.0001. NHS, normal human serum; Zymosan (5 μg/mL); ApoE, recombinant human apolipoprotein E (ε3 isoform from Novoprotein, 10 and 3 μg/mL in A and B resp.); A1AT, alpha-1-antitrypsin (10 μg/mL).

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Complement deposition on ApoE was assayed using NHS. (A-D) C1q-, C4b-, C3b-, and C9 deposition using classical complement deposition conditions (DGVB++ buffer) on immobilized recombinant ApoE and plasma derived ApoE. (E, F) C1q and C4b deposition on different immobilized ApoE isoforms. Positive control h.a. IgG was coated at 1 μg/mL in A and E, and 2.5 μg/mL in B, C, D, and F; ApoE was coated at 10 μg/mL, and A1AT at 10 μg/mL. For each panel, mean ± SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s multiple-comparisons test was applied to determine statistically significant differences between plasma-derived and recombinant ApoE or between ApoE isoforms. No statistically significant differences were found for all panels. NHS, normal human serum; IgG, heat-aggregated IgG; ApoE_R, recombinant human Apolipoprotein E of the ε3 isoform from Novoprotein; ApoE_P, plasma-derived Apolipoprotein E; A1AT, alpha-1-antitrypsin; Apo E2, recombinant human Apolipoprotein E of isoform ε2 from Athens Research; Apo E3, recombinant human Apolipoprotein E of isoform ε3 from Athens Research; Apo E4, recombinant human Apolipoprotein E of isoform ε4 from Athens Research.

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Recombinant, Clinical Proteomics, Derivative Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: We analyzed inhibition of C4b and sC5b-9 deposition on positive control (label ‘C’ on x-axis, coated heat-aggregated IgG, 2.5 μg/mL; A, B), and inhibition of sheep erythrocyte lysis by ApoE (panel C), using recombinant ApoE of different isoforms and plasma-derived ApoE. Mean + SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and two-way ANOVA (A, C), and one-way ANOVA (B) with Dunnett’s post-tests were applied to determine statistically significant differences compared to the uninhibited controls (label ‘C’ in panel A and B, and ‘0 μg/mL’ conditions per protein in panel C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IgG, heat-aggregated IgG (2.5 μg/mL); ApoE, Apolipoprotein E (10 μg/mL in panel A); A1AT, alpha-1-antitrypsin (10 μg/mL); C, control on coated IgG, with amboceptor and without inhibitors; BSA, bovine serum albumin (10 μg/mL); C1-inh, C1-inhibitor (20 μg/mL); C4BP, C4b-binding protein; PRELP, Proline And Arginine Rich End Leucine Rich Repeat Protein (50 μg/mL).

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Inhibition, Positive Control, Lysis, Recombinant, Clinical Proteomics, Derivative Assay, Control, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein and BioPure, with (A, C) and without (B, D) gold-label and human plasma derived C1q were mixed in solution and adsorbed simultaneously to a dark stained surface to allow for negative imaging. In addition, ApoE with (F, H) and without (G, I) gold-label were allowed to adsorb to a dark stained surface, followed by adsorption of C1q. All experiments were performed once, and per condition 50 fields were evaluated. Quantification of mean + SD of at least 500 counted particles per field is depicted in panels E and J. Scale bar is 25 nm in panel I. Kruskal-Wallis test with Dunn’s post-test was applied to determine statistically significant differences between conditions, ****p<0.0001. N-terminal ApoE-Au, gold-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE-Au, gold-labeled ApoE bound to the C-terminal globular heads of C1q. N-terminal ApoE, non-labeled ApoE bound to the N-terminal stalk region of C1q; C-terminal ApoE, non-labeled ApoE bound to the C-terminal globular heads of C1q.

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Recombinant, Clinical Proteomics, Derivative Assay, Staining, Imaging, Adsorption, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Recombinant human ApoE of the ε3 isoform from Novoprotein was incorporated into PC/PE liposomes which were then coupled to streptavidin Dynabeads and complement deposition from NHS was assessed using primary antibodies against C1q (panel A) and C4b (panel B), followed by AF488-coupled secondary antibodies and mean fluorescence intensity was measured with flow cytometry. In addition, we tested the inhibitory capacity of ApoE when incorporated into liposomes, using a hemolytic assay of sheep erythrocytes. Flow cytometry data are normalized to empty liposomes incubated with 5% NHS as a basal value which was set to 100%, and mean + SD of 8 individual experiments are shown. Flow cytometry samples per experiment were analyzed in duplicate and two-way ANOVA with Tukey’s multiple-comparisons test was applied to determine statistically significant differences between empty liposomes and ApoE-carrying liposomes. Hemolytic assay was performed three times, mean + SD are plotted as percentage of full lysis, and one-way ANOVA with Dunnetts’s multiple-comparisons test was applied to determine statistically significant differences compared with normal lysis. Concentrations provided on y-axis labels are in μg/mL. NHS, normal human serum.

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Recombinant, Liposomes, Fluorescence, Flow Cytometry, Hemolytic Assay, Incubation, Lysis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Oxidation, citrullination, and carbamylation were induced on ApoE-coated Maxisorp plates followed by incubation with either protein solutions of C1q (A, B), incubation with NHS to assay C4b deposition in GVB++ buffer (C, D), or incubation with protein solutions of AF488-labeled C4BP (E, F). C1q binding was assayed using primary antibody and HRP-labeled secondary antibody and colorimetric assay, and C4BP binding was assayed using Alexa Fluor 488-prelabeled C4BP and fluorescence measurements. C1q binding, C4b deposition on ApoE_ox and ApoE_cit, and C4BP were normalized to 1 μg/mL IgG (A, B), to 1 μg/mL IgG at 2% NHS (C) or to 10 μg/mL C4met (E, F). Mean + SD (A, B, E, F) or ± SD (C, D) of three individual experiments are shown. Samples per experiment were analyzed in duplicate and one-way ANOVA with Dunnett’s (A, B, E, F) or two-way ANOVA with Sidak’s (C, D) multiple-comparisons test were applied to determine statistically significant differences compared to untreated ApoE. *p<0.05, **p<0.01, ****p<0.0001. IgG, heat-aggregated IgG; ApoE, recombinant human ApoE of the ε3 isoform from Novoprotein; ApoE_ox, oxidized ApoE; ApoE_cit, citrullinated ApoE; ApoE_carb, carbamylated ApoE; A1AT, alpha-1-antitrypsin. C4met, methylamine-treated C4.

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Incubation, Labeling, Binding Assay, Colorimetric Assay, Fluorescence, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: Oxidation, citrullination, and carbamylation were induced on ApoE-coated Maxisorp plates, followed by incubation with either NHS in Mg2+-EGTA buffer to assay C3b deposition (A, B), or with protein solutions of FH (C, D). Data per experiment were normalized to the positive controls (Zymosan for A and B; C3b for C and D), and are plotted as a percentage. FH binding was assayed using primary antibody and HRP-labeled secondary antibody and colorimetric assay. Mean + SD of three individual experiments are shown. Samples per experiment were analyzed in duplicate and one-way ANOVA with Dunnett’s multiple-comparisons test were applied to determine statistically significant differences compared to untreated ApoE. *p<0.05, **p<0.01, ****p<0.0001. ApoE, recombinant human ApoE of the ε3 isoform from Novoprotein (1 (A) and 10 μg/mL B-D)). ApoE_ox, oxidized ApoE; ApoE_cit, citrullinated ApoE; ApoE_carb, carbamylated ApoE; A1AT, alpha-1-antitrypsin (10 μg/mL). ApoE_ox, oxidized ApoE coated at 10 μg/mL; ApoE_cit, citrullinated ApoE coated at 10 μg/mL; ApoE_carb, carbamylated ApoE coated at 10 μg/mL; A1AT, alpha-1-antitrypsin (10 μg/mL), Zymosan (5 μg/mL), C3b (10 μg/mL).

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Incubation, Binding Assay, Labeling, Colorimetric Assay, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q

doi: 10.4049/jimmunol.1900372

Figure Lengend Snippet: A commercial ELISA was used for measuring ApoE (A), and an in-house developed ELISA (B) was used for measuring C4d in the SF of 30 RA patients. Assays were performed once, in single wells, to limit the amount of required patient material. ApoE and C4d concentrations were calculated using standard curves and plotted in ng/mL, with mean ± SD. Using the Spearman R test, no significant correlation was observed between C4d and ApoE in the SF of this cohort (C).

Article Snippet: ApoE isoforms ε2, ε3, and ε4 were purchased from AH Diagnostics Ab (BioPure, Biovision) and plasma-derived ApoE was purchased from Athens Research and Technology.

Techniques: Enzyme-linked Immunosorbent Assay